Different types of stem cells are being studied because of their capability of forming or regenerating organs and tissues when injuries occur. In contrast to embryonic stem cells, somatic stem cells can be recovered from autologous adult tissues, thereby decreasing the risk of graft rejection. Therefore, the isolation and characterization of stem cells from different tissues, such as bone marrow, adipose tissue, peripheral blood and umbilical cord (blood) are well known. However, the percentage of stem cells is very low in each organ or tissue, and therefore, also the purification is a very important aspect of stem cell research.
Since constant regeneration of the skin is achieved due to somatic stem cell differentiation within the epidermis and the hair follicle, skin may serve as an excellent source of epithelial stem cells (EpSCs) (Grandi et al., 2012; Staniszewska et al., 2011). Hence it has been proposed that EpSCs could be useful in the treatment of several diseases, such as burn wounds, chronic wounds, and ulcers (Draheim and Lyle, 2011). In addition, ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias could also benefit from these EpSCs (Draheim and Lyle, 2011).
Therefore, different attempts to sort EpSCs from mammal skin have been described (Fujimori et al., 2009; Nowak and Fuchs, 2009). Fujimori reported the isolation of human epidermal stem/progenitor cells by means of gravity assisted cell sorting. However, sorting cells based on their diameter is quite controversial, since there are a lot of non-stem cells with a small diameter as well. Nowak and Fuchs described fluorescence activated cell sorting (FACS) as a technique to isolate EpSCs from mice. Unfortunately, the cell sorting was performed based on a cell surface marker which is also present on other cell types. In both studies, no thorough characterization was carried out which could have confirmed the origin of the mixed cell populations.
Marcelo et al., 2012 describe a method for producing primary cell cultures from epidermal skin, oral mucosa and ureter by use of a trypsinization step. However, the cells obtained are primary keratinocytes, and hence have lost their multipotency, which makes them less attractive to use.
Recently, the isolation of equine cells with mammary stem/progenitor cell capacities was reported (Spaas et al., 2012) by making use of non-adherent plating conditions. However, the method disclosed herein has several lacunae, resulting in a mixed population of stem cells, their progeny and non-stem progenitor cells, as also previously described by Stingl in 2009. A similar conclusion can be drawn from the method for isolating and culturing bovine mammary epithelial stem cells, as described by Li et al. (2008).
EP 1 414 947 equally described a method for obtaining highly pure populations of stem cell populations based on their property of being able to survive under non-adherent conditions. However, the obtained stem cell population only consisted of dermal stem cells, whereas it was found that no viable stem cells could be purified from the epidermis. Dermal stem cells are however restrictive to the regeneration of dermal cell types (e.g. adipocytes for fat tissue or fibroblasts for connective tissue). Epidermal stem cells are more advantageous when aiming to regenerate epidermal tissue, for instance as treatment for burning wounds or other skin damages (e.g. ulcers).
Researchers are currently also faced with problems identifying EpSCs, and especially from the differentiating EpSCs from MSCs. Although some markers are known (e.g. Spaas et al, 2012; Li et al., 2009; Marcelo et al., 2012; Juxue Li et al., 2008 and Sharifullina et al., 2005), none are found specifically suitable from distinguishing with certainty abovementioned cell populations. As the purity of an isolated cell population is crucial for downstream applications, adequate identification tools are a requisite.
Hence, there remains a need in the art for an improved method for isolation of a pure population of epithelial stem cells, preferably yielding a pure population of stem cells from epidermal origin as well as to provide an adequate marker set to identify the EpSCs. The invention thereto aims to provide an optimized method for isolating, purifying and characterizing EpSCs derived from mammalian skin, which can for instance be used for cosmetic and/or therapeutic purpose.